Phytochemical Compounds and Antioxidant Activity of the Pulp of Two Brazilian Passion Fruit Species: Passiflora Cincinnata Mast. And Passiflora Edulis Sims

ABSTRACT About 600 species of Passiflora are described around the world, of which 120 are native to Brazil. In the present work, the phytochemical compounds were analyzed by high-performance liquid chromatography, and the antioxidant activities (AOX) were measured in the pulp of P. edulis and P. cincinnata species. The P. edulis presented a higher bioactive content and AOX when compared to P. cincinnata. Citric and malic acids were the main organic acids found. The principal component analysis-PCA associated the highest values of quercetin-3-glucoside, hesperidin, trans-caftaric acid, chlorogenic acid, caffeic acid, p-coumaric acid, trans-resveratrol, rutin, procyanidin-B2, epigallocatechin-gallate and epicatechin-gallate, to P. cincinnata. The P. edulis was associated with higher values of total phenolic, vitamin C, total carotenoids, AOX (DPPH•, ABTS+• and FRAP), cis-resveratrol, naringenin, kaempferol-3-glucoside, myricetin and procyanidin-B1. P. cincinnata, although not a commercial species such as P. edulis, presented phenolic compounds and antioxidant activity in acceptable values, including the high values of quercetin 3-glucoside. The present study contributes to the knowledge of the physical-chemical composition of the Passiflora species.


Introduction
Passion fruit is a tropical fruit belonging to the family Passifloraceae. The name was given by Spanish missionaries to South America who used the exotic flower to describe and explain their religious convictions while trying to convert the indigenous habitants to Christianity. The most well-known types of this fruit are the purple and yellow passion fruits, Passiflora edulis Sims (Wijeratnam, 2016).
Around the world, approximately 600 species of Passiflora are described, of which 120 are native to Brazil (Ayres et al., 2017). Among Brazilian passion fruit species, yellow passion fruit (Passiflora edulis Sims) is the most cultivated and appreciated due to its flavor and aroma (Rotili et al., 2013). Besides in Brazil, P. edulis species have also been cultivated with economic significance in countries such as Ecuador, Colombia, Peru, Indonesia, and Kenya. The yellow passion fruit represents over 95% of the production for juice extraction (Wijeratnam, 2016).
The genus Passiflora is traditionally used in the treatment of anxiety and as an antidepressant. Yellow passion fruit leaf extracts (P. edulis) have been shown to have antidepressant activity in rats similar to the effects of the commercial drugs Nortriptyline and Fluoxetine; this effect has been attributed to its phytochemical compounds, such as flavonoids (Ayres et al., 2017). Seed extracts from P. edulis have also shown antitumor activity and antioxidant activity associated with fatty acids present in this seed (Mota et al., 2018). Peels of P. edulis are rich in pectin and their flours can be used to replace some colloids used in the production of processed foods (Coelho et al., 2018a(Coelho et al., , 2016. The passion fruit species Passiflora cincinnata Mast. popularly known in Brazil as "maracujádo-mato", "maracujá-da-caatinga", "maracujá-mochila" or "maracujá-tubarão" is native to the Caatinga, an exclusive biome of Brazil (Lavor et al., 2018). Passiflora cincinnata Mast., synonyms: Passiflora corumbaensis Barb. Rodr., Passiflora perlobata Killip or Passiflora cincinnata var. minor Hoehne, is a species of plant in the Passifloraceae family belonging to the Passiflora genus. It is vigorous plant of grooved stems. Its leaves are palmate, digitate, with 3-5 lobes, split ones. The flowers, with 10 cm of diameter, have roughened and curled cilia on corona filaments, ringed or snail-shaped with hair, white striped and alternate purple striped on the base. The fruit is ovoid, with 5 × 3 cm, and seeds measuring 0.5 cm long (Costa et al., 2015). The fruit has a greenish rind and white pulp, having a higher acidity content than P. edulis. The P. cincinnata is a species resistant to pests and agricultural diseases and has been used in the genetic improvement of new varieties of passion fruit (Siebra et al., 2018), such as cv. 'BRS Sertão Forte' (P. cincinnata), recently created by the Brazilian Agricultural Research Corporation (Embrapa) breeding program (Silva et al., 2020). It has also been used in the production of handmade jams and juices (Azoubel et al., 2011). Extracts from the leaves, peel, and seeds of P. cincinnata have been shown to have in vitro antimicrobial and anti-inflammatory activities, with these activities being attributed to its phenolic compounds (Lavor et al., 2018;Siebra et al., 2018).

Raw Material
The passion fruits P. cincinnata and P. edulis were harvested at the experimental farm of the Brazilian Agricultural Research Corporation (EMBRAPA Semiarid), Petrolina, PE, Brazil (09º09ʹ S and 40º22ʹ W). The average rainfall of the region is 1.78 mm, concentrated in the period from January to April in 2016, the year of the study. The average monthly temperature was 26°C, relative humidity between 50 and 70%, average insolation of 2800 hours/year, with evaporation around 2000 mm/year. The soil of the harvested area is red-yellow Latosol type. The fruits were sanitized in chlorine solution at 50 ppm, cut, and the pulp was manually extracted, packed in polyethylene bags, and kept in a freezer (−20°C) until analysis. For each species of passion fruit, three replications were analyzed, where each replicate corresponded to the mixed pulp of 30 fruits randomly collected from 15 plants.

Classical Analysis and Color Measurement by CIEL*a*b* System
To obtain basic analytical characteristics of the samples, classical pH analyses (potentiometer B 474 Micronal (Brazil)) were carried out; soluble solids (ºBrix) (digital refractometer HI 96801 Hanna, USA), titratable acidity (TA), humidity, and ashes, following the methodologies described in (AOAC, 1998). Color analysis was also conducted using a MiniScan EZ digital colorimeter (Hunterlab, USA) and CIE Lab L*, a* and b* system, where L* denotes lightness and ranges from 0 (black) to 100 (white), and a* and b* denote opponent dimensions, ranging from green (-) to red (+) and from blue (-) to yellow (+), respectively.

Total Bioactive Content -Total Phenolic Content, Total Carotenoids, and Vitamin C
The total phenolic content of the samples was measured by the colorimetric method with Folin-Ciocalteu according to Singleton and Rossi (1965). Gallic acid was used as standard and the total phenolic concentrations in samples were expressed as mg of gallic acid equivalents (GAE)/kg. One gram of fresh pulp was mixed with 4 mL of methanol 50% v/v using vortex for 2 min. The mixture was centrifuged (4000 × g 5 min, 24°C) and the supernatant was removed. One aliquot of the extract was used for quantification of the total phenolic content and assays of the antioxidant activity.
Total carotenoids were determined according to the method described in AOAC (1998). The extraction of the carotenoids was by liquid-liquid extraction with hexane as the organic phase. Fifty mL of ultrapure water, 10 g of passion fruit pulp, 30 mL of isopropanol and 10 mL of hexane were added to a separatory funnel and the mixture was stirred and allowed to stand for 30 min. The organic phases of two extractions were mixed, 0.2 g of sodium sulfate, 5 mL of acetone was added, and the volume was topped up to 50 mL with hexane. The absorbance was read at 450 nm in the spectrophotometer, and the results expressed in mg of β-carotene per kg of the fresh pulp.
The vitamin C (ascorbic acid) was determined according to Ramaiya et al. (2013), using a direct titration method. Ascorbic acid was used as the standard. The passion fruit pulp (25 mL) was titrated against iodine solution until the endpoint blue-black color developed.

Simultaneous Analysis of Organic Acids and Sugars by HPLC-DAD-RID
Simultaneous analyses of the organic acids and sugars were performed using an HPLC system model Agilent 1260 Infinity LC (Agilent Technologies, Santa Clara, CA, USA) coupled to a DAD-Diode Arrangement Detector (G1315D model) and RID-Refractive Index Detector (G1362A model). This procedure was carried out according to the chromatographic conditions previously described by Coelho et al. (2018b). A 500 μL aliquot of fresh pulp was diluted in 4500 μL of ultrapure water, filtered through a 0.45 μm nylon membrane (Chromafil® Xtra, Macherey-Nagel -Germany), and a volume of 10 μL was injected into a column. The column used was that of Agilent Hi-Plex H (300 × 7.7 mm) ion exchange with internal particles of 8.0 μm protected by a PL Hi-Plex H (5 × 3 mm) guard column (Agilent Technologies). The temperature of the column compartment was maintained at 70°C, and RID flow cell at 50°C. The flow used was 0.5 mL/min with a run time of 20 min. The phase was H 2 SO 4 4.0 mmol L −1 . For the determination of tartaric, malic, lactic, succinic, citric, and acetic acids, detection was made in the DAD at 210 nm. For glucose and fructose sugars, detection was made in the RID. Data were processed using OpenLAB CDS ChemStation Edition TM software (Agilent Technologies, USA).
The ABTS+• radical was formed through the reaction of 7 mM ABTS in 140 mM potassium persulfate for 16 hr in the absence of light. The solution was then diluted in ethanol until absorbance reached 0.700 ± 0.05. The ABTS +• radical scavenging activity of the samples was determined through the rate of decay in the absorbance at 734 nm determined at time t = 0 min and at time t = 6 min after the addition of samples.
The DPPH • method of the samples was assessed through the rate of decay in the absorbance at 517 nm. A solution of DPPH • (1.0 mmol L −1 ) was prepared in ethanol and diluted to an absorbance of 0.900 ± 0.050 (100 µmol L −1 ). The absorbance of the DPPH • solution was determined at time t = 0 min and 30 min after the addition of sample.

Statistical Analysis
All the analyses were carried out in triplicate and the results were expressed as average ±standard deviation of the values obtained. To evaluate the behavior of the phenolic profile, total carotenoids, vitamin C and antioxidant activity in relation to the Passiflora species, the application of multivariate statistics was carried out using principal component analysis (PCA) with pretreatment of the data for normalization and scaling. The program SPSS Inc., version 17.0 (Chicago, IL, USA) was utilized.

Basic Analysis and Color
The results of the classic analysis and color of the passion fruit pulp from the species P. edulis and P. cincinnata are shown in Table 1. The values of pH, Brix degree (ºBrix) and titratable acidity (TA) in P. edulis pulp were 2.87, 12.23 and 6.28%, respectively. In the P. cincinnata pulp, the values were 2.74, 13.16 and 8.41% for pH, ºBrix and TA, respectively. In general, the results obtained for pH and ºBrix showed few differences between the two passion fruit species evaluated. The values of TA in passion fruit P. cincinnata was higher than in P. edulis.
The values of pH, ºBrix and TA found in the present study for passion fruit P. edulis agree with other studies that have analyzed the pulp of passion fruit from several other species, such as P. edulis (Purple), P. edulis (Frederick), P. maliformes, P. quadrangularis, P. edulis (Pink) and P. edulis Flavicarpa. In these species, pH values varied from 2.6 to 3.54, TA of 1.19-5.5% and ºBrix of 10.1-16.8 (Chen et al., 2018;Dias et al., 2011;Ramaiya et al., 2013;Silva et al., 2000). Based on these values, the titratable acidity of passion fruit P. cincinnata (8.41%) was considered high.
Brazilian legislation establishes that commercial pulp from the passion fruit must have a pH between 2.7 and 3.8, and ºBrix greater than 11 (Brasil, 2000). Based on this, the values of pH and The results are expressed as mean ± standard deviation (n = 3). ND -not detected. L* = lightness, a* = redgreen hue, b* = yellow-blue hue.
ºBrix of the two evaluated species meet the requirements of Brazilian legislation for the commercialization of pulp. The values of humidity and total ash in P. edulis pulp were 85.95 and 0.98%, respectively. For the P. cincinnata pulp, the humidity and total ash presented values of 85.15 and 0.81%, respectively. In the pulp of Passiflora foetida, 76% humidity content and 1.7% total ash have been reported (Song et al., 2018).
Regarding the color measured by the CIE L*a*b* system, the pulp of the passion fruit P. edulis presented values of L* = 62.27, a* = 15.65 and b* = 97.63. The pulp of passion fruit P. cincinnata showed values of L* = 75.35, a* = −2.1 and b* = 26.02. Considering that positive values of b* corresponding to yellow, and negative values of a* corresponding to green, it can be observed that in the pulp of P. edulis the yellow color predominates, and in P. cincinnata there was a slightly greenish color, as it can see in Figure 1. The color of the pulp of passion fruit species is usually associated with the presence of compounds such as anthocyanins, carotenoids, and yellow flavonoids (Silva et al., 2014;Wondracek et al., 2011).

Organic Acids and Sugars
The values obtained for organic acids and sugars from the fresh pulp of the passion fruit species P. edulis and P. cincinnata are shown in Table 1. The major organic acids quantified in the pulp were citric and malic acids, and succinic acid in small amounts. The values of citric and malic acids in the pulp of P. edulis were 3.87 and 1.60 g 100 g −1 , respectively. In the P. cincinnata pulp, the values of citric and malic acid were 6.85 and 1.66 g 100 g −1 , respectively. The values of succinic acid were 0.08 and 0.17 g 100 g −1 in the pulp of P. edulis and P. cincinnata, respectively. The profile and values of organic acids in fruit pulp depend on many factors, such as type of fruit, variety, and maturation stage. Citric acid with values ranging from 0.03 to 5.15 g 100 g −1 and malic acid (0.01-2.18 g 100 g −1 ) are the main organic acids present in the pulp of various fruits such as melon, cashew, açai, peach, orange and lemon (Flores, Hellín & Phenol, 2012;Scherer et al., 2008). In the yellow passion fruit pulp P. edulis flavicarpa and the passion fruit P. foetida, citric and malic acids were also the main detected organic acids (Oliveira et al., 2014;Song et al., 2018). Based on the values obtained for organic acids in the present study, the pulp from the passion fruit species P. edulis and P. cincinnata were found to be high in acidity, mainly because of the high amount of citric acid. Regarding sugars, the values obtained for glucose and fructose in the pulp of P. edulis were 1.27 and 0.01 g 100 g-1, respectively. The P. cincinnata pulp had values of 0.11 and 0.02 g 100 g-1 for glucose and fructose, respectively. Glucose and fructose are the main sugars present in the passion fruit pulp of several species such as P. edulis (yellow), P. edulis (Purple), P. edulis (Frederick), P. maliformes, P. quadrangularis, P. edulis (pink) and P. edulis flavicarpa (Ramaiya et al., 2013). The glucose and fructose values in the passion fruit pulp used in the present study agree with those obtained by Song et al. (2018), in passion fruit pulp P. foetida.

Total Bioactive Content -Total Phenolics, Total Carotenoids and Vitamin C
A number of studies have associated the bioactivity content of passion fruit fresh pulp with phenolic compounds, carotenoids, and ascorbic acid (Medina et al., 2017;Pertuzzati et al., 2015;Ramaiya et al., 2013;Song et al., 2018;Wondracek et al., 2011).
The results for total phenolics measured with Folin-Ciocalteu, total carotenoids and vitamin C (ascorbic acid) obtained in the fresh pulp from P. edulis and P. cincinnata are presented in Table  2. Total phenolics presented values of 365 and 476.1 mg kg −1 for P. cincinnata and P. edulis, respectively. For total carotenoids, expressed as β-carotene, the values were 18.7 and 0.9 mg kg −1 for the P. edulis and P. cincinnata, respectively. Ascorbic acid values were 264.2 and 176.1 mg kg −1 for the pulp of P. edulis and P. cincinnata, respectively. Studies evaluating the total phenolic content of passion fruit pulp by the Folin-Ciocalteu method have reported values ranging from 277 to 600 mg kg −1 in the P. edulis (yellow), P. edulis (purple), P. edulis (Frederick), P. maliformes, P. quadrangularis, P. edulis (pink), P. edulis flavicarpa and P. glandulosa Cav. species (Chen et al., 2018;Ramaiya et al., 2013;Song et al., 2018). For vitamin C (L-ascorbic acid) in several Passiflora species, values varying from 23 to 577 mg kg −1 are described (Pertuzatti et al., 2015;Ramaiya et al., 2013;Rotili et al., 2013). Based on this, the total phenolic and vitamin C contents of the pulp of P. edulis and P. cincinnata were considered to be within normality. The content of carotenoids present in passion fruit pulp depends on many variables, including the crop system, climatic factors, and fruit maturation (Pertuzatti et al., 2015;Vianna-Silva et al., 2008). Wondracek et al. (2011) evaluated 14 individual carotenoids in the pulp of four Brazilian passion fruit species (P. nitida, P. cincinnata, P. setacea and P. edulis) and mentioned that the trans-β-carotene was present in all species, with cis-ζ-carotene being the main carotenoid found in P. edulis (6.2-12.1 mg kg −1 ), followed by trans-ζ-carotene (5.4-12.1 mg kg −1 ) and trans-β-carotene (2.6-3.8 mg kg −1 ), all associated with the yellow color of the pulp. Concerning P. cincinnata, only trans-β-carotene (0.03 mg kg −1 ) and violaxanthin (0.02 mg kg −1 ) were present in small amounts. In a study by Pertuzatti et al. (2015), 5 individual carotenoids from yellow passion fruit pulp P. edulis harvested from organic and conventional crop systems were evaluated, where βcryptoxanthin (139-250 mg kg −1 ) was the main quantified compound, followed by β -carotene (0.6-0.8 mg kg −1 ).
In the present work, the pulp of the yellow passion fruit P edulis Sims. presented higher values of total carotenoids (18.7 mg kg −1 ) than P. cincinnata (0.9 mg kg −1 ), corroborating the study by Wondracek et al. (2009).

Individual Phenolic Compounds
The profiles of phenolic compounds quantified by HPLC analysis of the passion fruit fresh pulp studied are shown in Table 2. Among the 21 phenolic compounds analyzed, 7 were flavanols, 4 flavonols, 2 flavanones, 6 phenolic acids and 2 stilbenes. Chromatograms obtained from the analysis of phenolic compounds by RP-HPLC-DAD of the extracts (LLE) of the pulp of P. edulis and P. cincinnata can be observed in Figure 2a,b, respectively.
In relation to the quantified flavanones, the pulp of P. edulis presented values of 0.46 and 2.74 mg kg −1 for naringenin and hesperidin, respectively (Table 2). P. cincinnata presented values of 0.14 and 4.58 mg kg −1 for naringenin and hesperidin, respectively.
Few studies have analyzed hesperidin and naringenin in passion fruit pulp. Shanmugam et al. (2018) evaluated the Brazilian passion fruit pulp P. subpeltata and did not find naringenin. Usually naringenin and hesperidin are the major phenolic compounds in citrus fruits such as oranges, lemons, tangerines and grapefruit, reaching values of 1162 and 1674 mg kg −1 , respectively (Zhao et al., 2017).

Non-flavonoids: Phenolic Acids and Stilbenes
The results obtained for phenolic acids and stilbenes in passion fruit fresh pulp are presented in Table  2. The values of trans-resveratrol and cis-resveratrol in the pulp of P. edulis were 0.22 and 2.32 mg kg −1 respectively; and in P. cincinnata pulp, these were 0.24 and 1.64 mg kg −1, respectively. Trans and cis resveratrol stilbenes have been widely studied for their health benefits to consumers, where grapes and grape-derived beverages have been the main source studied for these compounds. The values obtained for these stilbenes in the pulp of P. edulis and P. cincinnata agree with those mentioned in the literature for juices and wines cultivated in the same region as the studied passion fruit (Dutra et al., 2018); this confers merit on this species as bioactive compounds of nutritional interest.
In relation to phenolic acids, the main compounds present in terms of quantity were caffeic acid, chlorogenic acid and syringic acid in values from 1.06 to 1.74 mg kg −1 . Gallic acid, ρ-coumaric acid and trans-caftaric acid, with values ranging from 0.30 to 1.42 mg kg −1 were also detected. In a study by Rotta et al. (2019), the pulp of P. edulis presented hydroxybenzoic acid (0.12 mg kg −1 ), chlorogenic acid (0.18 mg kg −1 ), vanillic acid (0.43 mg kg −1 ), caffeic acid (0.06 mg kg −1 ), coumaric acid (0.24 mg kg −1 ) and trans-cinnamic acid (0.37 mg kg −1 ). For the pulp of passion fruit P. subpeltata, they detected the presence of cinnamic acid (0.01 mg kg −1 ), ferulic acid (0.05 mg kg −1 ) and vanillic acid (0.05 mg kg −1 ). The values obtained for phenolic acids in the pulp of P. edulis and P. cincinnata in the present work are higher than reported in other studies for passion fruit pulp P. edulis (Rotta et al., 2019) and P. subpeltata (Shanmugam et al., 2018); however, these compounds were present in small amounts in both studies.

In Vitro Antioxidant Activity
In Figure 3, the results of the antioxidant activities (AOX) of the passion fruit fresh pulp as measured with ABTS +• and DPPH • are expressed as equivalent to Trolox per kilogram of fresh pulp (mmol TE kg −1 ). The results of the AOX by the FRAP method are expressed as Fe 2+ equivalent per kilogram of fresh pulp (mmol Fe 2+ kg −1 ). With the free radical sequestration methods, P. edulis pulp had mean values of 1.47 and 1.43 mmol TE kg −1 for ABTS +• and DPPH • , respectively. P. cincinnata pulp had mean values of 1.25 and 1.07 mmol TE kg −1 for ABTS +• and DPPH • , respectively. With the FRAP method, the pulp of P. edulis and P. cincinnata presented average values of 5.92 and 4.50, respectively. In general, the pulp of passion fruit P. edulis had higher values of AOX than P. cincinnata.
The antioxidant activity of a sample depends on the method used in the measurement as well as the composition of the sample in relation to the profile of antioxidant substances. In the present study, except for the high quercetin 3-glucoside values in P. cincinnata, the two types of pulp studied presented a close profile for phenolic compounds; however, the highest values of AOX in P. edulis may be associated with its higher total bioactive content -total phenolics, total carotenoids and ascorbic acid (see Table 2). Other works in the literature have also associated the total phenolic content of passion fruit pulp directly to the AOX analysis by DPPH • and ABTS +• methods (Ramaiya et al., 2013;Rotta et al., 2019;Septembre-Malaterre et al., 2016).
Based on the results of PCA, the differences in chemical profiles between the two species of passion fruit studied are evident. The species that presented the highest antioxidant activity was P. edulis, and, using by PCA, the compounds strongly correlated with antioxidant activities (DPPH • , ABTS +• and FRAP) had the highest values of total phenolics, total carotenoids and vitamin C (ascorbic acid) present in the pulp of this species of passion fruit ( Table 2). The results obtained in the present work corroborate with other studies that have mentioned that phenolic compounds, carotenoids and ascorbic acid are the main bioactive compounds present in the pulp of Passiflora (Medina et al., 2017;Pertuzatti et al., 2015;Ramaiya et al., 2013;Rotta et al., 2019;Wondracek et al., 2011).
The PCA proved to be a technique capable of differentiating the two species of passion fruit by virtue of their chemical profiles, even when the differences in the values of the measured compounds were apparently close (see Table 2). P. cincinnata, although not a commercial species such as P. edulis, presented phenolic compounds and antioxidant activity in acceptable values, especially the high values of quercetin 3-glucoside. The present study highlights that P. cincinnata is a Brazilian native species that needs to be better explored. The results of the phenolic profile obtained in the present study for P. cincinnata Mast, corroborate those obtained recently by Silva et al. (2020) for the cv. 'BRS Sertão Forte', variety resulting from the genetic improvement of the species Passiflora cincinnata Mast, where quercetin 3-glucoside (isoquercetin) was the main phenolic found.